earticle

영어

In eukaryotic cells, organellar proteome biogenesis is pivotal for cellular function. Chloroplasts contain a complex proteome, the biogenesis of which includes post-translational import of nuclear-encoded proteins. Protein-targeting machineries must provide chaperones to protect their membrane-protein substrates from aggregation and to keep them in a translocation-competent state. However, the mechanisms determining when and how nascent chloroplast-targeted membrane proteins are sorted and guided in the cytosol are unknown. Here, we establish the timing and mode of interaction between ankyrin repeat-containing protein 2 (AKR2A), the cytosolic targeting factor of chloroplast outer membrane (COM) proteins, and its interacting partners during translation at the single-molecule level. The targeting signal of a nascent AKR2A client protein residing in the ribosomal exit tunnel induces AKR2A binding to ribosomal RPL23A. Subsequently, RPL23A-bound AKR2A binds to the targeting signal when it becomes exposed from ribosomes. AKR2A-mediated targeting of COM proteins is coupled to their translation, which in turn is crucial for biogenesis of the entire chloroplast proteome. On the other hand, structural analysis, molecular modeling, and mutational analysis of the Ankyrin repeat domain (ARD) of AKR2A identified two adjacent sites for coincidental and synergistic binding of monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG), and this and this binding is very important for the membrane insertion or import into the chloroplast of their cargo proteins. Finally, based on the understanding of the targeting mechanism of these membrane proteins, we investigate the possibility of utilizing organelles as oral delivery vehicle for protein therapuetics such as vaccines and protein drugs through engineering of the chloroplast outer membrane and membrane proteins.